IGF-I and insulin regulate glucose transport in mouse blastocysts via IGF-I receptor

The roles of glucose deprivation, insulin, and insulin-like growth factor I (IGF-I) in the regulation of glucose transport in the mouse blastocyst were examined. Glucose transport, measured by uptake of 3-O-methyl glucose (3-OMG), was increased by 19% (P < 0.01) in response to glucose deprivation. Both IGF-I and insulin stimulated uptake, but IGF-I was 1,000-fold more potent than insulin, increasing uptake by 51% at 1.7 pM (P < 0.001). These effects began to appear after 20 min of incubation with growth factors, and required the simultaneous presence of glucose. The relative potencies of insulin and IGF-I suggest that the actions of IGF-I and insulin were both mediated via the IGF-I receptor. The inactivity of a specific agonistic insulin receptor antibody (B10) confirms this and suggests that this action may be independent of signalling through IRS-1. Cycloheximide decreased growth factor-stimulated transport by about 40%, indicating that both protein synthesis and transporter recruitment from cytoplasmic stores are responsible for maximal stimulation. These characteristics are consistent with GLUT1-facilitated glucose uptake and suggest that GLUT1 is the regulatable transporter in mouse blastocysts. Stimulation of GLUT1 may be a ubiquitous feature of the autocrine/paracrine activity of IGF-I in cell growth and proliferation. © 1996 Wiley-Liss, Inc.     

Human formyl peptide receptor function role of conserved and nonconserved charged residues.

In this study, we investigated the role of charged residues in ligand binding interactions of f-Met-Leu-Phe receptors (FPR). Charged residues of FPR, both conserved and nonconserved, which are located close to the membrane interface were mutated to alanine to determine their role in ligand binding. The mutated residues belonged to specific domains of FPR which have previously been implicated in FPR ligand binding interactions. We demonstrate that nonconserved charged residues such as Arg84, Lys85, Arg205 and Asp284 and conserved charge residue Arg163 seem to play a role in ligand binding. However, alteration of nonconserved charged residue Asp106 did not have any effect. In conclusion, specific charged residues of FPR, both conserved nonconserved, may contribute to FPR function either directly or indirectly.     

Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison.

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150-200 and 1421-1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.     

Rapid detection of conserved regions in protein sequences using wavelets

We present an algorithm to detect protein sub-structural motifs from primary sequence. The input to the algorithm is a set of aligned multiple protein sequences. It uses wavelet transforms to decompose protein sequences represented numerically by different indices (such as polarity, accessible surface area or electron-ion integration potentials of the amino acids). The numerical representation of a protein sequence has significant correlation with its biological activity, thus common motifs are expected to be observable from the wavelet spectrum. The decomposed signals are then up-sampled and similarity search techniques are used to identify similar regions across all the proteins at multiple scales. Results indicate that wavelet transform techniques are a promising approach for rapid motif detection.     

Insulin and IGF-I signaling through the insulin receptor substrate 1

The insulin and insulin-like growth factor-I (IGF-I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF-I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of ?160 kD, now known as the insulin receptor substrate 1 (IRS-1). We have purified IRS-1 from mouse 3T3-L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS-1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) binds tightly to the activated platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3-kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3-L1 adipocytes, a portion of the Ptyr form of IRS-1 becomes tightly complexed with PI 3-kinase. Since IRS-1 binds to fusion proteins containing the SH2 domains of PI 3-kinase, association most likely occurs through this domain. The association of IRS-1 with PI 3-kinase activates the enzyme about fivefold. Thus, one signaling pathway from the insulin and IGF-I receptors probably proceeds as follows: tyrosine phosphorylation of IRS-1, tight association of IRS-1 with PI 3-kinase with accompanying activation of the kinase, elevation of the PI 3-phosphates. © 1993 Wiley-Liss, Inc.     

Comparison of anti-apoptotic signalling by the insulin receptor and IGF-I receptor in preadipocytes and adipocytes

We compared the effectiveness of insulin receptor (IR) and type I insulin-like growth factor (IGF) receptor (IGFR) cytoplasmic domains in mediating anti-apoptotic effects in 3T3-L1 preadipocytes and adipocytes. We used TrkC/IR and TrkC/IGFR chimeras, stably expressed in these cells and stimulated with neurotrophin-3 (NT-3), so as to avoid interference from endogenous receptors. After 24-h serum deprivation, 10% of preadipocytes and 2% of adipocytes appeared apoptotic as determined by fluorescence-activated cell sorter (FACS) analysis of cells stained with propidium iodide (PI) and Annexin V. When NT-3 was added, the two chimeras inhibited apoptosis to the same extent by 80% in preadipocytes and 50% in adipocytes. Mutation of juxtamembrane tyrosines (IR Y960F, IGFR Y950F) abrogated these anti-apoptotic effects. Qualitatively similar results were obtained by determination of viable rather than apoptotic cells. We conclude that IR and IGFR have equal potential to inhibit apoptosis in cell backgrounds, which are normally responsive to either IGF-I or insulin.     

IGF-I and insulin receptor signal transduction in trout muscle cells

In this study, primary cultures of trout skeletal muscle cells were used to investigate the main signal transduction pathways of insulin and IGF-I receptors in rainbow trout muscle. At different stages of in vitro development (myoblasts on day 1, myocytes on day 4, and fully developed myotubes on day 11), we detected in these cells the presence of immunoreactivity against ERK 1/2 MAPK and Akt/PKB proteins, components of the MAPK and the phosphatidylinositol 3-kinase-Akt pathways, respectively, two of the main intracellular transduction pathways for insulin and IGF-I receptors. Both insulin and IGF-I activated both pathways, although the latter provoked higher immunoreactivity of phosphorylated MAPKs and Akt proteins. At every stage, increases in total MAPK immunoreactivity levels were observed when cells were stimulated with IGF-I or insulin, while total Akt immunoreactivity levels changed little under stimulation of peptides. Total Akt and total MAPK levels increased as skeletal muscle cells differentiated in culture. Moreover, when cells were incubated with IGF-I or insulin, MAPK-P immunoreactivity levels showed greater increases over the basal levels on days 1 and 4, with no effect observed on day 11. Although Akt-P immunoreactivity displayed improved responses on days 1 and 4 as well, a stimulatory effect was still observed on day 11. In addition, the present study demonstrates that purified trout insulin receptors possess higher phosphorylative activity per unit of receptor than IGF-I receptors. In conclusion, these results indicate that trout skeletal muscle culture is a suitable model to study the insulin and IGF-I signal transduction molecules and that there is a different regulation of MAPK and Akt pathways depending on the developmental stage of the muscle cells.     

Prediction of both conserved and nonconserved microRNA targets in animals

MOTIVATION: MicroRNAs (miRNAs) are involved in many diverse biological processes and they may potentially regulate the functions of thousands of genes. However, one major issue in miRNA studies is the lack of bioinformatics programs to accurately predict miRNA targets. Animal miRNAs have limited sequence complementarity to their gene targets, which makes it challenging to build target prediction models with high specificity. RESULTS: Here we present a new miRNA target prediction program based on support vector machines (SVMs) and a large microarray training dataset. By systematically analyzing public microarray data, we have identified statistically significant features that are important to target downregulation. Heterogeneous prediction features have been non-linearly integrated in an SVM machine learning framework for the training of our target prediction model, MirTarget2. About half of the predicted miRNA target sites in human are not conserved in other organisms. Our prediction algorithm has been validated with independent experimental data for its improved performance on predicting a large number of miRNA down-regulated gene targets. AVAILABILITY: All the predicted targets were imported into an online database miRDB, which is freely accessible at http://mirdb.org.     

Comparison of several protein sequences by recognition of conserved regions

The sequences of related proteins show the alternance of conserved and variable regions. This fact is generally seen as a reverberation of 3 D constraints onto 1 D structures. Although the exact meaning of such constraints remains elusive, conserved regions can be extracted from protein chains and used to align them. We developed a program that efficiently performs this task. The program constructs symbolic motifs fitting a target subsequence present in every chain without requiring any insertion or deletion. However, a motif can be obliterated by substitutions when it is found in a sequence. The motifs formally consist in aminoacid symbols separated (and virtually preceded and followed) by a variable number of wild-card symbols. A wild-card, which can match any aminoacid of the chains (with no increment of score), represents a variable site within conserved regions. Different motifs are progressively built by substituting a wild-card with an aminoacid symbol within or beside preexisting motifs. Only those motifs showing an outstanding association of high matching score over all chains, and of low deviation between extreme scores over individual chains are selected for making the next generation. Starting with a null motif, the construction ends when no new aminoacid can be introduced into the current motifs. A surviving motif is then considered valid if it maps without ambiguity a unique region in every sequence, and the motif with highest score is finally selected. The construction of new motifs is then reinitated for the left and right parts of the sequences, after these have been split by the previously selected motif.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.     

Differential roles of the insulin and insulin-like growth factor-I (IGF-I) receptors in response to insulin and IGF-I

Insulin and insulin-like growth factor-I (IGF-I) receptors are highly homologous tyrosine kinase receptors that share many common steps in their signaling pathways and have ligands that can bind to either receptor with differing affinities. To define precisely the signaling specific to the insulin receptor (IR) or the IGF-I receptor, we have generated brown preadipocyte cell lines that lack either receptor (insulin receptor knockout (IRKO) or insulin-like growth factor receptor knockout (IGFRKO)). Control preadipocytes expressed fewer insulin receptors than IGF-I receptors (20,000 versus 60,000), but during differentiation, insulin receptor levels increased so that mature adipocytes expressed slightly more insulin receptors than IGF-I receptors (120,000 versus 100,000). In these cells, insulin stimulated IR homodimer phosphorylation, whereas IGF-I activated both IGF-I receptor homodimers and hybrid receptors. Insulin-stimulated IRS-1 phosphorylation was significantly impaired in IRKO cells but was surprisingly elevated in IGFRKO cells. IRS-2 phosphorylation was unchanged in either cell line upon insulin stimulation. IGF-I-dependent phosphorylation of IRS-1 and IRS-2 was ablated in IGFRKO cells but not in IRKO cells. In control cells, both insulin and IGF-I produced a dose-dependent increase in phosphorylated Akt and MAPK, although IGF-I elicited a stronger response at an equivalent dose. In IRKO cells, the insulin-dependent increase in phospho-Akt was completely abolished at the lowest dose and reached only 20% of the control stimulation at 10 nm. Most interestingly, the response to IGF-I was also impaired at low doses, suggesting that IR is required for both insulin- and IGF-I-dependent phosphorylation of Akt. Most surprisingly, insulin- or IGF-I-dependent phosphorylation of MAPK was unaltered in either receptor-deficient cell line. Taken together, these results indicate that the insulin and IGF-I receptors contribute distinct signals to common downstream components in response to both insulin and IGF-I.     

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

The evolution of signal receptor proteins: conserved regions and the similarity to GTP-binding proteins

A sequence comparison of signal receptor proteins (SR) was carried out using computer techniques based on physicochemical characteristics of amino acids. A new method of conserved regions determination for a family of proteins is described. Visual pigments have four, and all SR--three such regions in the cytoplasmic loops. Possible functional significance of these regions is discussed. We also report here that the family of SR is similar with the family of G-proteins involved in extracellular signal transduction. Both families have similar regions consisting of 7-8 amino acids and a number of identical amino acids distributed on the considerable part of the polypeptide chain of the proteins. These facts may indicate that the whole ensemble of the proteins participating in transmembrane signalling pathways (or some part of it) could evolve from a common progenitor. At the same time, similar structure elements of members of the mentioned protein families my be functionally important for protein-protein interaction.     

TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus.

The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.     

IGF-I and INS receptor expression in the salivary glands of diabetic Nod mice submitted to long-term insulin treatment

This work aimed to characterize the IGF-I and INS receptor expression in the salivary glands of Nod mice, correlating to therapeutic effects of insulin treatment on these receptors. Nod mice were divided into: Groups 1 and 2 (diabetic), Groups 3 and 4 (diabetic with insulin treatment) and Group 5 (non-diabetic). Fragments from the salivary glands were processed for immunohistochemical analysis. The results showed that the prolonged diabetic state led to a steadily increased IGF-I receptor expression. INS receptor expression was gradually decreased. It was concluded that not only was the IGF-I receptor expression affected by the diabetic state but also the INS receptor expression. The period of the diabetic state was directly related to changes in the expression of these receptors. In spite of the insulin treatment having recovered the glycaemic levels, the expression of INS and the IGF-I receptors did not reach the standard level, which certainly hampered glandular function.     

Use of protein knobs to characterize the position of conserved alpha-subunit regions in lutropin receptor complexes

Efforts to identify the manner in which human choriogonadotropin (hCG) contacts lutropin receptors (LHR) have been stymied by the complex structure of the hormone and the likelihood that it contacts the receptor at multiple sites. During studies of hCG assembly in mammalian cells, we found that addition of a cysteine to the long disordered beta-subunit COOH terminus (betaCT) enabled it to become cross-linked by a disulfide to cysteines that are substituted for residues in loop alpha2 or in the alpha-subunit COOH terminus (alphaCT). This created a "knob" on the alpha-subunit at the location of the cysteine. Knobs of various sizes and charges were useful for probing surfaces of the alpha-subunit thought previously to contact the LHR. Attachment of the betaCT to residues in loop alpha2 facing loops beta1 and beta3 reduced hormone activity only a few fold revealing that this surface does not participate in essential high affinity receptor contacts, a finding inconsistent with our earlier view of the hCG-LHR complex. In contrast, this approach showed that the opposite surface of loop alpha2 appeared to be nearer the receptor interface. Although attachment of knobs to portions of the alphaCT reduced hormone activity substantially, this finding was difficult to interpret. As discussed, this procedure should be adapted readily to other proteins and may facilitate the introduction of fluorophores, enzymes, or other reagents at specific sites on protein surfaces. It may also permit one to cross-link proteins or to obscure specific protein surfaces during the development of "Trojan Horse" therapeutics.     

High affinity antibody from hen's eggs directed against the human insulin receptor and the human IGF-I receptor

Large quantities of high affinity antibodies directed against the human insulin receptor and the human insulin-like growth factor-I (IGF-I) receptor were obtained from hen's eggs. Hens were immunized with human placental membranes and human liver membranes by intramuscular injections. Specific antibodies to the receptors were demonstrated in serum and egg yolks at 5 weeks and these antibodies presisted for at least 6 months. Antibodies from egg yolks were purified by the polyethylene glycol precipitation technique of Polson et al. The eggs provided the equivalent of about 450 ml of immunized serum per month per bird. The purified antibodies were approximately equally reactive with receptors for insulin or IGF-I. Antibodies immunoprecipitated affinity-labeled receptors, inhibited binding of each ligand, and were capable of stimulating 2-deoxyglucose uptake in rat adipocytes and thymidine incorporation in cultured fibroblasts. The presence of antibodies directed against the IGF-I receptor in those hens immunized with human liver membranes was unexpected, since liver membranes possess little or no IGF-I binding activity. We conclude that antibodies against human antigens may be relatively easily obtained by immunization of hens and purification of those antibodies from eggs.